Melanoma is a common cancer of the skin resulting in high morbidity and mortality. Melanomas are malignancies of melanocytes, the specialized pigment cells of the skin, located at the basal layer of the epidermis and which originate from neural crest. Melanoma is one of the most aggressive cancer types in human. Melanoma accounts for only about 4% of skin cancer cases but for as many as 74% of all skin cancer deaths. In 2002, the WHO estimated 160,000 new cases of malignant melanoma worldwide and reported 41,000 deaths caused by this dreadful disease (Parkin D. M. et al., Cancer J. Clin. 55: 74, 2005). It is the cancer type with the highest increase in incidence: of all cancer in the United States, cutaneous melanoma ranks fifth in incidence among men and seventh among women and is the second leading cause of lost productive years. Recent estimates suggest a doubling of melanoma incidence every 10-20 years (Garbe C. and Leiter U., Clin. Dermatol. 27: 3, 2009). If melanoma can be diagnosed early, it can be cured by surgical excision and this is what occurs in appr. 80% of the cases. However, metastatic melanoma is refractory to current therapies and has a very poor prognosis with a median survival rate of 6 months. Both due to the high propensity to metastisize as well as resistance to available therapies, melanoma represents a great problem for oncology.
Several genes have been implicated in the development of melanoma. The most common tumor suppressor gene involved in melanoma is p161nk4a, encoded by the CDKN2A locus. The CDKN2A locus on human chromosome 9p21 encodes two proteins, p16ink4a and p14ARF, that mainly regulate cell cycle progression and cell survival via the pRb and p53 pathways, respectively. Loss of p16 is accomplished through deletion, mutation or promoter methylation. Mutations in the p14ARF tumor suppressor gene also play a role in melanoma, independent of the effect of the p16ink4a gene. The most commonly mutated oncogenes in melanoma are BRAF and N-RAS (Q61K/R), which are generally mutually exclusive. Interestingly, BRAF is mutated in ˜70% of malignant melanomas, papillary thyroid cancer (36-53%), serous ovarian cancer (˜30%) and colorectal cancer (5-22%), of which the majority is the V600E mutation. In addition, other BRAF mutations have also been detected in, serous ovarian cancer (30%) and lung cancer (3%) (Garnett M. J. et al., Cancer Cell, 2004). However, there are in at least 35 other amino acids within the BRAF protein that are targets for mutations in melanoma (Dhomen N. et al., Hematol. Oncol. Clin. North Am., 23: 529, 2009). The V600E mutation results in constitutively active BRAF and has been shown to act as an oncogene in melanocytes. As a consequence of the somatic mutations of BRAF and N-RAS, the RAS-RAF-MEK-ERK MAPK signal transduction pathway, that controls a variety of biological responses, including proliferation and survival, is constitutively active. The aberrant activation of this pathway results in increased proliferation and survival, but also represents an attractive molecular target for melanoma treatment. The importance of MAPK activation in melanoma was shown by inhibiting BRAF with RNAi and inhibiting BRAF or MEK with small molecule inhibitors (Hingorani S. R. et al., Cancer Res. 63: 5198, 2003, Karasarides M. et al., Oncogene 23: 6292, 2004). Such treatments block cell proliferation, survival, induce apoptosis and inhibit anchorage independent growth. Additional pathways that are aberrantly activated in melanoma are the PI3K/PTEN/Akt pathways. The phosphoinositide-3-kinase (PI3K) and mitogen-activated protein (MAP) kinase pathways are two key signaling cascades that have been found to play prominent roles in melanoma development. Therefore, members of the PI3K signaling pathway may also function as interesting targets for therapeutic intervention (Madhunapantula S. V. et al., Pigment Cell Melanoma Res. 22:400-19, 2009).
At present, enormous efforts are taken to unravel the molecular mechanisms that lead to changes in cellular processes and the resulting malignant behaviour of transformed melanocytes. One family of molecules involved in the genesis and progression of melanoma cells, the miRNAs, is currently attracting a lot of attention.
miRNAs are naturally occurring single-stranded, non-coding small RNA molecules that control gene expression by binding to complementary sequences in their target mRNAs, thereby inhibiting translation or inducing mRNA degradation. miRNAs have recently emerged as key regulators of gene expression during development and are frequently misexpressed in human disease states, in particular cancer.
Recently, several groups have taken a miRNA profiling approach, in which melanoma cell lines and/or melanoma samples were used to identify miRNA signatures and/or miRNAs that might play a regulatory role in melanoma. Some studies correlated expression of particular miRNAs with survival (Caramuta S. et al., J. Inv. Dermatol., 2010, Satzger I. et al., Int. J. Cancer 126: 2553, 2009 and Segura M. et al., Clin. Cancer Res. 16: 1577, 2010), mutational status (Caramuta S. et al.), progression (Mueller D. et al., J. Inv. Dermatol. 129: 1740, 2009, Segura M. et al., PNAS 106: 1814, 2009), chromosomal aberrations (Zhang L. et al., PNAS 103: 9136, 2006, Segura et al, 2009, Radhakrishnan A. et al., Mol. Vis. 15:2146, 2009) or merely described differential expression of specific miRNAs (Ma Z. et al., J. Mol. Diagn. 11:420, 2009, Stark M. et al., Plos One 5: e9685, 2010, Jukic D. et al., J. Transl. Med. 8: 27, 2010, Philippidou D. et al., Cancer Res. 70: 4163, 2010). Others identified signatures of miRNAs to distinghuis between different tissue types (Gaur A. et al., Cancer Res. 67: 2456, 2007, Blower P. et al., Mol. Cancer. Ther. 6: 1483, 2007, Lu J. et al., Nature 435: 834, 2005) or different stages of development (Radhakrishnan A et al., Caramuta S. et al.)
None of the above mentioned studies characterised melanoma-specific miRNAs in depth. The challenge is to relate specific miRNAs to their cellular function, and to unravel the impact of specific miRNAs in the formation and progression of malignant melanoma.
There is a limited amount of studies that have looked at specific miRNAs in melanoma. Some profiling studies ultimately resulted in focus on individual miRNAs and their function. Schultz et al. performed expression analysis of nevi and melanoma samples and focused on the Let-7 family, which was downregulated in melanoma. Overexpression of Let-7b in melanoma cells downregulated the expression of Cyclin D1, D3 and A and Cdk4, and consequently resulted in inhibition of cell cycle progression and anchorage independent growth (Schulz J. et al., Cell. Res. 18: 549, 2008). A similar profiling study comparing nevi and metastatic melanoma by Chen et al. resulted in identification of miR-193b downregulation in metastatic melanoma. They showed that reintroduction of miR-193b reduced proliferation and G1 arrest through regulation of Cyclin D1 (Chen J. et al., Am. J. Pathol. 176: 2520, 2010).
In uveal melanoma, miR-34a expression was found to be diminished. Reintroduction of miR-34a resulted in decreased proliferation and migration. c-Met as a target for miR-34a was suggested to be involved (Yan D. et al., Inv. Ophtamol. 50: 1559, 2009)
Other studies determined whether known melanoma specific genes are regulated by miRNAs. MITF, a transcription factor involved in melanocyte development was shown to be regulated by miR-340 (Goswami S. et al., J. Biol. Chem. 285: 20532, 2010), miR-137 (Bemis L. et al., Cancer Res. 68: 1362, 2008) as well as miR-182 (Segura M. et al., 2009). The latter also regulates FOXO3 and functional experiments showed that expression of miR-182 enhanced migration of melanoma cells and metastatic potential. Similarly, HOXB7, a transcription factor involved in melanoma, was found to be regulated by miR-196a Inhibition of miR-196a resulted in HOXB7, its downstream target bFGF and ultimately an increased migration of a melanoma cell line. Additionally, 2 studies demonstrated that c-Kit, a receptor enhancing the tumorigenic potential of transformed melanocytes, is regulated by miR-221 and 222. Overexpression of miR-221/222 resulted in increased proliferation, migration and anchorage-independent growth in vitro and enhanced tumor growth in vivo (Igoucheva O. et al., Biochem. Biophys. Res. Com., 2009 and Felicetti F. et al., Cancer Res., 2008). Lastly, target prediction programs predicted that Integrin B3 may be regulated by Let-7a. Integrin β3 is known to play an important role in melanoma progression and invasion and its expression is increased during melanoma progression. Expression of Let-7a shows an inverse correlation in melanoma cell lines. Let-7a was able to regulate Integrin B3 and its inhibition resulted in increased migration (Muller D. et al., Oncogene 27: 6698, 2008)
Most of the studies that identified miRNAs involved with melanoma are profiling studies and/or were focussing on a specific miRNA. Most studies did not select a given miRNA based on a comparative functional analysis of a library comprising more than 1000 miRNAs. There is still a need for identifying miRNAs involved in melanoma using a functional screen, wherein the miRNA affects proliferation, apoptosis, survival, invasion and/or migration.
There is currently no effective known medicament that may be used for specifically preventing, treating, regressing, curing and/or delaying a disease or condition associated with melanoma or for diseases or conditions associated with activated BRAF pathway in a subject. The only standard treatments comprise chemotherapy, radiotherapy, surgery. Therefore, there is still a need for diagnostic markers for melanoma and for new treatments of disease or conditions associated with melanoma.